The invention relates to a wave field microscope having an illumination or excitation system.
By using highly specific labels, such as DNA probes or protein probes, it is possible to label virtually arbitrarily small (sub)structures, in biological (micro-)objects, especially in cells, nuclei, cell organelle, or on chromosomes,—referred to in the following simply as objects. Structures can be specifically represented in dimensions of a few μm (10−6 m) up to a few tens of nm (10−9 m). The labels are usually coupled to fluorochromes, or also to colloidal (gold) micro-particles, to facilitate their optical detection and image formation, i.e., to render them possible in the first place.
To be able to detect two labels within the same object, separately from one another, the labels in question are often coupled to heterochromatic fluorochromes. The available color emission spectrum of the fluorochromes usually used ranges from deep blue, through green, red, and up to the infrared spectral range. However, fluorochromes can also be used, which are neither differentiated in their excitation spectrum, nor in their fluorescence spectrum, but whose fluorescence emission lifetime is used as a distinguishing parameter.
The advantage of the latter is that wavelength-dependent focal shifts do not occur. Fluorochromes can also have different emission spectra and, thus, varying spectral signatures, but be excitable by the same photon energy, e.g., by multiphoton processes. Here as well, one can avoid wavelength-dependent focal shifts in the excitation between fluorochromes having different spectral signatures.
The aforementioned fluorochromes that are able to be or are bound to specific (substructures in biological micro-objects are designated in the following as fluorescence labels.
If the excitation spectra and/or emission spectra and/or the fluorescence lifetimes of two fluorescence labels match, then these fluorescence labels have the same spectral signature with respect to the parameter in question. If the fluorescence labels differ in one or more parameters relevant to the measurement, then they have different spectral signatures.
Fluorescence is understood in the following to be any photon interaction, in which differences arise between the excitation spectrum and the emission spectrum of the same substance that are not attributable to monochromatic absorption or dispersion. This also includes, in particular, multiphoton interactions, in which the excitation wavelengths can be greater than the emission wavelengths.
The concept of fluorescence is used here as well for the narrowly related phenomena of luminescence, in particular for phosphorescence. This includes, in particular, longer average fluorescence lifetimes, e.g., fluorescence lifetimes in the range of up to several or many msec (milliseconds). The closely related processes of luminescence, phosphorescence, and fluorescence are considered in the following as having equal relevance to the present invention.
Fluorescence labels in spatially extended biological objects are detected, imaged, and quantitatively localized with respect to defined object points/object structures (distance and angular measurements) using light-microscopic measuring methods. A decisive role is played in this connection by the so-called “point spread function”=PSF or “point response” of the microscope used, or generally of the optical system, i.e., its ability to construct from an “ideal punctiform” object, an equally ideal punctiform image. The point spread function is a characteristic feature of every imaging optical system, and a measure of its quality.
Distance measurements between object structures depend substantially on the effective point spread function—i.e., that given locally in the labeled object point. This effective point spread function, in turn, is considerably dependent on the specific local refractive index and the absorption in the object, in the object's embedding medium, in the immersion fluid and, in some instances, in the cover slips.
Generally, the effective point spread function clearly differs from the point spread function calculated for the microscope employed. As a rule, the point spread functions measured under technically optimized marginal conditions also differ from the effective point spread functions attainable in biological objects under practical, routine laboratory conditions.
Since, for the most part, these effective point spread functions are not available, to calibrate distance measurements, one reverts to ideal, calculated results or to calibration measurements performed under typical conditions, such as reflection methods. However, both methods are detrimental to precision in the case of three-dimensional distance measurements in biological micro-objects. Consequently, there is considerable uncertainty in determining the actual spatial distance between the object structures. In the case of biological objects, such quantitative size estimations contain uncertainties of up to several micrometers.
Up until recently, the virtually unanimous conviction prevailed in the scientific community that two object structures can only be separated if they are spaced apart by at least the width at half maximum intensity of the effective point spread function.
It was not until 1996 that the originators of the present invention succeeded in devising a calibration method which is not necessarily prior art to the present application for distant field microscopy (and also flow fluorometry), making it possible for high-precision distance measurements to be made between object structures, which are spaced apart by a distance smaller than the resolution of the distant field microscope in question, i.e., smaller than the width at half maximum intensity of the effective point spread function, independently of the position of the object structures in question in the three-dimensional space.
This method includes the following steps:                Before, during, or after preparing the object in question on or in an object holder, in particular a slide, object carrier fiber, object carrier capillary tube, or object carrier fluid, the structures (measuring structures) to be examined or to be localized are labeled with fluorescent stains having different and/or the same spectral signatures, i.e., such structures to be localized (measuring structures) directly proximate to one another, namely within the width at half maximum intensity of their effective point spread function, are labeled with fluorescent stains having different spectral signatures, while such measuring structures, whose distance from one another is greater than the width at half maximum intensity of the effective point spread function, are labeled with fluorescent stains having different or the same spectral signatures. Two measuring structures to be localized may then always be labeled with the same spectral signature, when they can be clearly identified, for example, by their relative position or by other criteria.        Calibration targets of a defined size and spatial arrangement are labeled with the same fluorescent stains;        the fluorescing calibration targets are either prepared together with the objects, or separately on or in the/an object holder (slide, object carrier fiber, object carrier capillary tube, object carrier fluid, or the like).        The (specimen) object and calibration targets are examined under identical conditions, simultaneously or sequentially, microscopically or flow-fluorometrically.        Two defined calibration targets having different spectral signatures are measured at a time under consideration of the wavelength-dependent imaging and localization properties of the particular optical system (microscope or flow-fluorometer). The measured values ascertained in the process, equivalent to the actual values, are compared to the previously known, actual distance values, equivalent to the reference values (i.e., to the reference localizations calculated on the basis of the geometry), and the difference between the actual values and reference values, namely the calibration value, is used to correct the shift, which is conditional upon the optical system, in the detection of various emission loci, in particular of the measuring structures.        
In other words: the distance measurement is performed between the object (sub)structures labeled (depending on the proximity to one another) with different or same spectral signatures—in the following, also referred to as measuring structures—on the basis of the highly precise localization of independent (calibration) targets having corresponding spectral signatures and having known sizes and spatial configurations, under consideration of the wavelength-dependent imaging and localization properties of the particular optical system, the calibration measurements taking place between the (calibration) targets, and the measurements taking place in the biological objects, under the same system and marginal conditions. These calibration targets have the same or a higher multispectral quality than do the (object) structures to be measured. They can be arranged directly in the biological objects or be present as separate preparations on an object holder (slide or object carrier fiber/capillary tube or object carrier fluid, or the like), or be part of an object holder. One can discriminate between two or more fluorescing measuring structures in intact, three-dimensional biological objects, whose spacing and spatial extent is smaller than the width at half maximum intensity of the effective point spread function, on the basis of their differing spectral signatures (fluorescence-absorption wavelengths and/or fluorescence-emission wavelengths and/or the fluorescence-emission lifetimes), i.e., one can determine the distances between them.
The distance measurement is reduced to the localization of the individual structures to be measured and can be performed—at this point, using optical distant field microscopy or flow fluorometry, as well—with a substantially higher precision than the width at half maximum intensity of the point spread function. The localization of the point of concentration of the measuring structures in question is adapted to the maximal intensity of their fluorescence signal. This means that, from the measured (diffraction-limited) signal (=intensity curve) of a fluorescent point (=fluorescing measuring structure),—under consideration of the composite information from the primary and secondary maxima—the point of concentration (bary center) of the signal is determined and, thus, the location of the measuring structure. When working with optical systems that are free from defects and, consequently, with ideal symmetry of the measured intensity distribution (=characteristic of the intensity curve), the point of concentration (bary center) of the intensity curve colocalizes, within the localization accuracy, with the primary maximum (=maximum 0 order of the diffraction image) of the measured intensity distribution.
With this new calibration method, optical distant field microscopy, such as wave field microscopy (or also scanning flow fluorometry) can be used to measure geometric distances in biological micro-objects, whereby the distances to be determined can be smaller than the width at half maximum intensity of the effective point spread function in the object. Since the information content of the distance determinations performed therewith corresponds to a distance measurement obtained at an increased resolution, one can (and will in the following) also speak in abbreviated form of “resolution equivalent”.
Using multispectral calibration, one can perform in situ measurements at the specific biological object, on the basis of the system's imaging properties. When the fluorescence lifetime is used as the sole parameter type and/or the fluorochromes are excited with the same photon energy (energies), the calibration eliminates the need for in situ correction of the chromatic shift in the object plane. This calibration method renders possible three-dimensional, geometric distance measurements in biological objects, all the way down to a level of molecular precision (i.e., resolution equivalent better then 10 nm), for the highest resolving distant field microscope types, such as the wave field microscope, and given the use of suitable fluorescence labels.
To determine the actual and reference values, for comparison thereof, and to define the correction value/calibration value, the following method steps are preferably carried out:                one or a plurality of calibration targets B having a distance greater than the width at half maximum intensity of the effective point spread function from the point of concentration of the N measuring structures is/are labeled with any desired spectral signature;        the distances dik (i, k=1 . . . N, i≠k) of the points of concentration of the spectrally separated diffraction figures of the N measuring structures, and the distances diB of the N measuring structures from the calibration target B are measured, automated methods for image analysis being applied;        for one measuring structure, the segments dik and diB are each measured in the plane of the narrowest point spread function, as are remaining distances, for which the object is rotated in each instance axial-tomographically by a defined angle Φm;        optical aberrations from the calibration measurements are corrected and, in each case, a cosine function Aik cos (φm+θik) or AiB cos (φm+θiB) having suitable phase shift is adapted to the corrected measured distances dik (θm) and diB (θm);        the maxima Aik and AiB of the adaptation function of dik or diB are divided by the magnification factor and determined as the Euclidian distance Dik or DiB of the N measuring structures, from one another, or of the distances of the measuring structures to reference point B.        
To determine the maxima, one preferably draws additionally on the corresponding minima of the distance zik, ziB in the plane orthogonal to the plane of dik, diB, and subjects them to analog analysis.
All coordinates of the N measuring structures and their relative coordinates to reference point B, i.e., positions xi, yi, zi and xk, yk, zk, or distances xk−xi, yk−yi, zk−zi and xB−xi, yB−yi, zB−zi are determined in accordance with the present invention on the basis of the microscopically measured 3D distances Dik or DiB, preferably using the following system of equationsD2ik=(xk−xi)2+(yk−yi)2+(zk−zi)2 D2iB=(xB−xi)2+(yB−yi)2+(zB−zi)2 D2kB=(zB−xk)2+(yB−yk)2+(zB−zk)2 
To guarantee the ascertained measuring results, the procedure described above should be carried out for a plurality of calibration targets B and for the same N measuring structures.
The coordinates and distances of the N measuring structures can be determined on the basis of the points of concentration, which are derived from the barycentric averages of the measurements for all reference points.
For graphical representations in particular, the ascertained positions xi, yi, zi and xB, yB, zB preferably undergo convolution using a point spread function, whose half width is that of the resolution equivalent achieved in each instance.
For the fluorochrome labeling of measuring structures and of calibration targets, preferably those fluorochromes are used which can be excited in the ultraviolet, visible and/or infrared light wavelength range, and which emit in the ultraviolet, visible and/or infrared light wavelength range. As calibration targets, one can use either biological calibration targets or non-biological, i.e., synthetic calibration targets.
The biological calibration targets are labeled regions of the biological object whose proximity to one another is known. The region(s) in question can, for example, be labeled using suitable biochemical probes. The practical advantage of using such biological calibration targets over synthetic calibration targets, for example calibration spherules, is that in performing the calibration, besides the optical marginal conditions of the object, marginal effects that are conditional upon the specimen also enter into the calibration, such as the actual fluorescence signal's relationship to the non-specific background (which is determined by automatic image analysis algorithms).
Especially suited as non-biological, i.e., synthetic calibration targets are micro-spherules, which have the same or a higher multispectral signature than the measuring structures to be localized. They are handled in the same way as the biological objects.
Calibration targets of this kind are preferably fixed to object holders in a defined spatial arrangement. This can be done already at the time that the slide in question is fabricated, which is particularly advantageous for routine use. To rectify the problem encountered with all known distant field microscopy methods, that the width at half maximum intensity of the point spread function and, thus, the resolution limit is dependent upon the relative position in the space, i.e., for example, normal to the optical axis (=lateral) it is narrower than in the direction of the optical axis (=axial), the mentioned calibration method can be easily combined with the so-called micro-axial tomography methods known in the related art. In these micro-axial tomography methods, the (biological) objects are arranged in capillary tubes or on glass fibers and in, i.e., under the microscope, definably rotated about an axis, which is usually normal to the optical axis of the microscope, distance measurements being carried out in that direction which has the narrowest width at half maximum intensity of the effective point spread function.
A distant-field light microscopy method which is particularly suited for detecting and imaging especially very small, fluorescently labeled substructures, in biological objects, is the wave field microscopy method. This method has the advantage over the known epifluorescence microscopy methods and or confocal laser scanning microscopy, that it renders possible depth discrimination—normal to the wave fronts—, in the axial direction as well. Thus, it makes it possible to have substantially improved resolution (at a higher numerical aperture, its dimensions can be substantially smaller than the wavelength of the light used for excitation).
In wave field microscopy, as described, for example, in U.S. Pat. No. 4,621,911, fluorescing, i.e., luminescing specimens are illuminated in the optical microscope by a standing wave field (standing wave field fluorescence microscopy, SWFM). A standing wave field is formed (only) where there is superposition of light that is capable of coherence. The specimens are arranged in a zone of equidistant, plane wave fronts, and excited to emit fluorescence or phosphorescence. The spacing of the wave fronts and their phases can be varied (in particular to produce images). The three-dimensional distribution of the fluorescent, i.e., luminescent object points can be reconstructed from the individual optical sections using computer-image processing.
The plane wave fronts are arranged normal to the optical axis of the detecting objective lens and are produced through coherent superposition of two laser beams at a defined angle q to the optical axis of the microscope system, the angle q defining the spacing of the wave fronts from one another—at a given wavelength and index of refraction. In place of two intersecting laser beams, the wave field can also be produced by forcing a laser beam, after suitable reflection, at a specific angle (for example, using a reflector), into interference with itself. The plane wave fronts are distinguished by the fact that the intensity profile is (co-) sinusoidal in the direction normal to the wave fronts.
The fluorescence, i.e., luminescence is either spectrally discriminated through suitable optical filters and conducted in various beam paths, or detected confocally. The attainable resolution, i.e., the smallest still measurable distance between two punctiform object structures, which are labeled by fluorochromes having the same spectral signature, is given either by the Abbe criterion (=the maximum 0 order of the diffraction image of a point object is localized in the 1st minimum of the diffraction image of a second point object) or is given by the width at half maximum intensity of the effective point spread function. It is dependent upon the particular wavelength, the numerical aperture of the objective lens employed, as well as upon the local refractive indices of the objects, of the embedding medium, of any cover slips used, and of any immersion fluids used.
In principle, the known wave field microscopes have the following design: they include    (I) an illumination, i.e., excitation system, made up of at least one real and one virtual illumination source, and at least one objective lens, so allocated to one another that they are able to produce a one-dimensional, sinusoidal, standing wave field;    (II) an object space, including holding and maneuvering devices for the object; and    (III) a detection system, made up of at least one objective lens, at least one eyepiece, and at least one detector, this often being a camera, in particular a CCD camera, which is positioned with the CCD chip in the intermediate image plane.
A drawback of this related-art wave field microscope, referred to in the following as “one-dimensional wave field microscope” (“SWFM”), i.e., of the wave field microscopy method that can be implemented with the microscope is that the periodically generated wave field (in the case of epifluorescent detection, in conjunction with optical sectioning methods) leads to an ambiguity in the observation or imaging of an object structure, whose extent in the direction normal to the wave fronts is substantially greater than λ/2n (λ=wavelength of the excitation, n=effective index of refraction). This ambiguity initially makes it more difficult to effectively benefit from the improved resolution achieved with the interference pattern.
To implement distance measurements and other examinations of the spatial relationships of three-dimensional objects, one can combine the known distant field microscopy methods, inclusive of one-dimensional wave field microscopy, with axial tomography. For this, the biological objects to be examined, in some instances after being furnished with calibration targets, are prepared as specimen in or on a micro-capillary tube or glass fiber, used as object holders or slides. The capillary tube/fiber has a precisely defined diameter, varying diameters being possible. To localize this capillary tube/fiber on the microscope table, a special mount fixture is proposed, which is made of a rigid, preferably dorsiventrally flattened frame, at or on which is mounted at least one bearing sleeve, in which a micro-capillary tube or glass fiber can be rotationally supported (preferably with the axis of rotation normal to the optical axis of the microscope). (The bearing sleeve(s) should be arranged in such a way that the axis of rotation of the capillary tube/fiber is normal the optical axis of the microscope.) The rotation of the specimen objects in or on the capillary tube/fiber follows directly from rotation of the capillary tube/fiber, preferably with the assistance of a torque motor.